The extracellular Ca2+ (Ca2+o) -sensing receptor (CaR) is a G- protein-coupled receptor (GPCR) that is a key mediator of direct effects of ca2+o on the function of parathyroid (PT) kidney and other cells. It plays a central role in normal ca2+o homeostasis and may also be involved in 1o ad 2o hyperparathyroidism (HPT). Therefore, a molecular understanding of how the CaR regulates cellular function would provide an important foundation for elucidating the physiology and pathophysiology of mineral metabolism. Several Ca2+o activated phospholipase are present in PT and other cells that could mediate the CaR's biological actions. It is not known which of these phospholipases are CaR-regulated, however, nor is it understood how the receptor activates some or all of them. That is, does it stimulate them directly via G-proteins or indirectly through intracellular messengers generated by primary, G-protein-regulated signaling pathways? Moreover, nothing is understood about the molecular determinants on the CaR's cytoplasmic domains that enable it to activate the G protein(s) regulating these effectors. Finally, little is known about the subcellular localization of the CaR and its spatial relationships to its intracellular effectors during activation. Preliminary studies for this proposal showed that the Car in PT cells is present within caveolae, specialized plasma membrane structures playing key roles in signal transduction initiated by cell surface receptors, including GPCRs, by promoting the formation of supramolecular complexes containing receptors, G-proteins and downstream effectors. Thus, caveolae could play an important role in PT cells by organizing key elements involved in signaling through this novel GPCR. The overall goal of this proposal is to determine the molecular basis, including the role of caveolae, for the coupling of the CaR to its associated G-proteins and, in turn, to key cellular signaling systems, including G-protein-mediated regulation of second messengers and secondary "cross-talk" with additional signaling pathways. The specific aims are: First, to test the hypothesis that vovine PT caveolae contain the mature, cell surface form of the CaR and other key elements in CaR-effector coupling and whether the CaR is resident within caveolae or recruited there upon CaR activation; second, to identify the G-protein(s) via which the CaR activates phospholipases; third, to characterize key intracellular domains of the CaR that couple it to G-protein; and finally, to document than the CaR mediates high Ca2+o elicited activating of PLC, PLA2+, and PLD as well as sphingomyelin turnover and to identify the mechanisms underlying regulating of these pathways. These studies should provide new insights into how the CaR regulates parathyroid function in health and disease states and, perhaps, into how it controls the function of other cells.